Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is inappropriately activated in many types of cancer, including multiple myeloma, leukemias, lymphomas, breast, head and neck, melanoma, ovarian, lung, pancreatic and prostate cancer. The role of STAT3 in promoting cancer cell proliferation and survival is well-established, and tumors and cancer cell lines with constitutive STAT3 activity have been shown to depend on STAT3 for survival. However, transcription factors have so far proven to be difficult to target with small molecule drugs because they lack hydrophobic pockets that can be bound by these drugs with high-affinity. With the exception of drugs targeting the nuclear hormone receptors, there has been little success in developing clinically effective inhibitors of transcription factors, including STAT3. Given these difficulties, an alternative strategy for targeting STAT3 is to identify other genes whose inhibition leads to cell death only in cells with active STAT3, a concept known as synthetic lethality or co-dependency. The proposed research aims to identify, using a panel of 100 human cancer cell lines of different lineages, co-dependencies with STAT3 activation. In order to identify STAT3-specific genetic dependencies, three markers of STAT3 activation were measured in a panel of 100 cell lines: phosphorylation at tyrosine 705, phosphorylation at serine 727, and ability to activate transcription using a luciferase reporter assay. Using these results, the cell lines were classifie as being STAT3-active or -inactive, then data from Project Achilles was used to identify genes required only in STAT3-active cell lines. Project Achilles is a large effort, initiated by the Hahn lab, in which genome-wide pooled shRNA screens are being performed in several hundred human cancer cell lines in order to identify essential genes in particular cancer cell types. This analysis identified approximately 230 genes as being STAT3 co-dependencies. The proposed research now aims to validate these candidate co- dependencies and to elucidate the mechanistic relationship between these genes and STAT3. The specific aims are: 1) to use CRISPR-Cas9 genome engineering to determine if STAT3-active cell lines depend on STAT3, 2) to validate candidate STAT3 co-dependencies, and 3) to investigate the mechanistic relationship between STAT3 and validated co-dependencies. While much research has focused on trying to identify novel STAT3 inhibitors, the research proposed here instead aims to identify co-dependencies with constitutive STAT3 activity and to elucidate the mechanistic basis for this interaction. The primary goal is to elucidate novel STAT3 biology and to obtain a more complete mechanistic understanding of STAT3 signaling. This work may also uncover potential drug targets for the many cancers that exhibit STAT3 activity.